Metalloproteinase oligopeptides and their therapeutic use

ABSTRACT

The invention discloses identification and therapeutic use of matrix metalloproteinase oligopeptides and peptidomimetics. The oligopeptides are used for making antibodies. The antibodies are used for diagnostic and treatment purposes of various diseases. In particular, the diseases may involve the mechanism of degradation of extracellular matrix by MMP&#39;s during cell proliferation cycle. Suppression of MMP activity seems to arrest tumor growth during cancer progression. MMP oligopeptides were used as vaccines to treat mice having murine melanoma B16FO induced tumor. There was a significant drop in tumor weight and size for the group of mice that were immunized with MMP oligopeptide.

This application contains sequence listing that has been submitted as anASCII file named RIPLLC018.003US1sequence_ST25.txt, the date of creationApr. 18, 2011, and the size of the ASCII text file in bytes is 2 kb.

FIELD OF TECHNOLOGY

This disclosure relates generally to designing and synthesizing novelmetalloproteinase oligopeptide sequences to be used as therapeuticagents for treating extracellular matrix related diseases. Morespecifically, this disclosure relates to using the metalloproteinaseoligopeptide as a vaccine and/or peptidomimetics to treat cancer.

BACKGROUND

Matrix Metalloproteinases are a family of zinc dependent neutralendopeptidases that play an important role in tumor angiogenesis, tissueremodeling, and cell migration. In cancer, levels of some MMP's areabnormally elevated, enabling cancer cells to degrade the extracellularmatrix (ECM), invade the vascular basement membrane, and metastasize todistant sites. A variety of pathological conditions are associated withan increased activity of metalloproteinases (MMP's), in particular MMP-2and MMP-9. These proteases are able to digest collagen and otherextracellular matrix (ECM) proteins as a precondition for the spreadingof the disease. Thus, there is a need for a therapeutic agent toeffectively block these MMP's from digesting the ECM, thereby blockingthe spread of cancer and other diseases.

Prevention and treatment of metastasis represents the major challenge incancer therapy today. The current available treatments are toxic,non-specific and unpredictable for ECM protein affected diseases. Thereis a need for a therapeutic agent to effectively block the MMP moleculesfrom digesting the ECM, thereby preventing ECM degradation and spreadingof diseases.

SUMMARY

The current disclosure discloses a sequence and a composition of MMPoligopeptide and a method of using the MMP oligopeptide as a vaccine anda peptidomimetic for treating ECM related diseases.

In one embodiment, the oligopeptide analogs for MMP-9 and MMP-2sequences were designed and synthesized. In another embodiment, theseoligopeptides were tested for their ability to inhibit cancer cellinvasion in-vitro using specific cell lines.

In another embodiment, the oligopeptide analogs of MMP-9 and MMP-2 weretested for their effectiveness for tumor growth suppression in-vivo inmice.

In one embodiment, the following oligopeptide sequences were used toproduce a vaccine.

MMP-9 Oligopeptides MMP-9 A1: -C-H-F-P-F-I-F-E-G-R-S- (SEQ ID NO: 2)    Y-S-A-C- MMP-9 A2: -D-T-D-D-R-F-G-F- (SEQ ID NO: 3) MMP-9A3: -D-R-D-K-L-F-G-F-C-P-T- (SEQ ID NO: 4)     R-A-D-S- MMP-2Oligopeptide MMP-2  A4: -C-P-R-K-P-K-W-D-K-C (SEQ ID NO: 1)

In one embodiment, the sequence of oligopeptide may but is not limitedto have mutations, deletions and substitutions.

In one embodiment, the MMP-2 oligopeptide may be used as a vaccineand/or a peptidomimetic. In another embodiment, MMP-9 oligopeptide maybe used in combination with any one of the MMP-2 oligopeptides as avaccine. In another embodiment, all four oligopeptide may be combined toproduce a vaccine.

The oligopeptide sequences, in one embodiment may be either linear orcircular in design. In another embodiment, the oligopeptide may repeatof sequences.

In another embodiment, the oligopeptide may have either haptens orpolyglycans attached to them for efficient delivery.

In another embodiment, a method of immunizing a mammal, such as mice, toraise antibodies for a specific MMP is disclosed. In one embodiment, aselection of an oligopeptide suitable for raising antigenicity isdisclosed. In another embodiment, inhibition of cancer cell invasion byoligopeptide induced antibody in-vitro was performed.

In another embodiment, mammals such as mice may be immunized and weresubsequently challenged with murine melanoma cancer cells B16F0 in orderto test the inhibition of tumor growth.

In one embodiment, the immunization of mammals may not be limited tocancer but may include all ECM degradation based disease treatment. Inanother embodiment, the vaccination may be done once or repeatedly bymeasuring the antibodies specific to the oligopeptide that was injected.

In one embodiment, a composition for an oligopeptide as a vaccine andpeptidomimetic comprising of oligopeptide MMP-9 A1, MMP-9 A2, MMP-9 A3and MMP-2 A4 individually or combination thereof.

In one embodiment the therapeutically effective amount may be rendered,but not limited to, as an injection. Other embodiments may includeperoral, topical, transmucosal, inhalation, targeted delivery andsustained release formulations.

The composition, method, and treatment disclosed herein may beimplemented in any means for achieving various aspects, and may beexecuted in a form suitable for the mammal. Other features will beapparent from the accompanying drawings and from the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Example embodiments are illustrated by way of example and not limitationin the figures of the accompanying drawings, in which like referencesindicate similar elements and in which:

FIG. 1 illustrates the prior art of MMP's digesting the ECM during adisease state.

FIG. 2 the method of treating a mammal using the vaccine.

FIG. 3 A-D shows the view of the matrigel invasion experiment in HeLacontrol cells as well as HeLa cervix cancer cells and the effect ofimmune sera from matrix metalloproteinase oligopeptide MMP-9 A 1-3 andMMP-2 A4.

FIG. 4 illustrates the weight of the mice before and after thetreatment.

FIG. 5 illustrates an effect of immunization using matrixmetallopeptidase MMP-9 A1-3 and MMP-2 A4 on tumor weight inhibition ofmelanoma cell line B16F0 xenografts in male C57BL/6 mice.

FIG. 6 is a view of the result tumor weight burden (length and width) onmale C57BL/6 mice before and after the melanoma xenograft treatment.

FIG. 7 A-E is a view of the tumor growth in control and tumor inhibitionin immunized mice.

FIG. 8 shows a view of the binding sited for the various peptidomimeticon a MMP molecule.

Other features of the present embodiments will be apparent from theaccompanying drawings and from the detailed description that follows.

DETAILED DESCRIPTION

Several sequences and methods for immunizing, treating cancer andreducing the size of the tumor using the matrix metalloproteinaseoligopeptide as a vaccine are described herein. Although the presentembodiments have been described with reference to specific exampleembodiments, it will be evident that various modifications and changesmay be made to these embodiments without departing from the broaderspirit and scope of the various embodiments.

Cancer cells produce higher levels of matrix metalloproteinases (MMP's),particularly MMP-9 and MMP-9. These enzymes are able to digest the extracellular matrix (ECM) connective tissue surrounding the cancerous cells.MMP's bind to ECM via specific binding sites. Blocking these bindingsites in the MMP's prevents the MMP's from binding to ECM. Inhibition ofECM destruction prevents the cancer progression and tumor sizereduction. In the current disclosure several potential binding siteswere identified within MMP-9 and MMP-2. FIG. 1 describes the cancerouscells 110 producing MMP's 120 (step 1). The MMP's 120 bind to thespecific binding sites at the ECM 130 (step 2). Step 3 in FIG. 1 showsthe MMP's 120 digesting the ECM 140.

FIG. 2 explains the current disclosure in steps of vaccine productionand immunization 200. Identification and design of the MMP's are carriedout 210 using the MMP sequences. Oligopeptides are synthesized 220 andrepresented by synthetic oligopeptides 240. The formulate oligopeptidesfor vaccination are 222. The antibodies are produced as shown in 226.The mice 242 are immunized 224 using vaccine with syringe 240. Theoligopeptides may be of specific length. Several permutations andcombinations of the sequences were tested. The instant disclosuresequences are shown in para [0007] as MMP-9 A1-3 and MMP-2 A4. Prior tothe selection of these four sequences as potential oligopeptides,several potential binding sites for MMP-9 and MMP-2 were identified. Atotal of eight oligopeptides were synthesized and tested usingfibrosarcoma HT-1080 cell line and cervical cancer cell line DoTC-2.Only four sequences were selected to be pursued. The MMP-9 A1-3 andMMP-2 A4 were synthesized in a linear and circular format. Manymodifications for these sequences were also done in one embodiment. Themodifications were substituting one or more amino acid residues atN-terminal, C-terminal and both C and N terminals, substitution of aminoacid residues based on similar charge and polarity, withoutconsideration of charge and polarity, omitting of amino acids in C and Nterminal, omitting only in C-terminal and only in N-terminal.

In another embodiment, substitution and omission may be carried outsimultaneously. The oligopeptides may be further modified by repeatingthe sequences and combining more than one MMP-9 A1-3 AND MMP-2 A4 forproducing and formulating a vaccine. The peptidomimetic to the MMP's maybe used to block the binding site of an overexpressed MMP in a specificdisease.

In one embodiment, the oligopeptide may be used as feedback regulatorsto specifically prevent or reduce the synthesis rate of MMP-9 and MMP-2productions at the cellular level. In one embodiment process of blockingand inhibition of ECM destruction by antigens produced due tovaccination of mice.

In another embodiment, the synthesized oligopeptides were biotinylatedat N-terminal using four carbon spacers by Genscript (Pitcataway, N.J.08554 USA) and conjugated to KLH protein. In an experiment performed onmice animals were procured, immunized, tumor induced and observed foreffectiveness of the treatment of oligopeptide-induced immunotherapy.

The injections were prepared using 100 μl l of KLH conjugatedbiotinylated peptides and 100 μl of complete Freund's adjuvant (Sigma,St. Louis, Mo.).

Male C57BL/6 mice 6 weeks of age on arrival were purchased from SimonsenLaboratories, Gilroy, Calif. and maintained in microisolator cage underpathogen-free conditions on a 12-h light/12-h dark schedule for a week.All animals were cared for in accordance with institutional guidelinesfor the care and use of experimental animals. After housing for a week,the mice (n=5/group) were immunized by intraperitoneal injection on Day0, and incomplete Freund adjuvant (Sigma) on Day 7, 14 and 28. The bloodsamples were tested for their immune response by standard Elisa testusing microtiter plates. Repeating injections of synthetic peptides inmice produce an immune response to specific individual peptides. Variousdilutions were tried and examples of dilutions tried are 1:100, 1:1000and 1:10000.

Conjugation of Peptides

All four peptides were conjugated covalently to keyhole limpethemocyanin (KLH) protein. In a typical experiment 1 mg of the peptide, 4mg of KLH was dissolved in 5 ml of 0.01 M NaHCO₃ and 2 μl ofglutaraldehyde and stirred overnight. At the end 100 μl of 1M glycineethyl ester to the final concentration of 0.1 M and left for 30 min atroom temperature. Then precipitated with 4-5 vol of cold acetone at −70C for 30 minute. Then briefly warmed at room temperature and pellet at10,000 g for 10 min. The pellet was dissolved in 1 ml of 0.1M NaHCO₃ andstored at −20° C.

Anti-Peptide Immune Response Assay

Microtiter plates were coated with individual peptide in 100 μl/well 5μg in mM carbonate buffer, pH 9.5 for 20 h at 20° C. After washing,wells were incubated with serial dilution of mouse serum in PBS/0.5%BSA, Tween™ 20 (binding buffer, BB). After washing, well were incubatedwith 100 μl/well rabbit anti-mouse IgG polyclonal antibody conjugatedwith horseradish peroxidase (HRP) in BB for 30 rain at 37° C. Afterwashing, the titer of anti-peptide mouse IgG was determined by colorreaction with TMB substrate solution at 450 nm.

At 1:100 dilution the intensity of immune response was higher for MMP-9A 1-3 and slightly less for MMP-2 A 4 as shown in Table 1 below.

TABLE 1 Elisa - A1-A4 peptide Non No No sp. pep Cont Test pep Test pepAvidin Peptide cox Sera #3 before B16FO given after B16FO given 1 2 3 45 6 7 8 9 10 11 12 CONT 1:100 .300 .166 .173 .352 .863 .241 .221 .267.862 1.345 .218 .202 1:1000 .149 .443 .158 .215 .339 .174 .164 .286 .699.234 .166 .164 1:10000 .119 .155 .154 .187 .261 .231 .212 .229 .287 .176.155 .148 MMP-9 A-1 1:100 1.248 .336 .362 .418 3.898 3.868 3.843 3.8953.897 3.864 3.820 3.87 1:1000 .417 .199 .299 .282 3.402 1.901 1.0332.127 3.624 1.875 .999 2.01 1:10000 .146 .210 .227 .244 .394 .274 .228.330 .645 .244 .226 .22 MMP-9 A-2 1:100 .927 .298 .299 .335 3.581 3.8633.870 3.238 3.635 3.609 3.465 3.86 1:1000 .208 .210 .189 .242 .646 1.3392.856 .360 .526 .859 .277 .274 1:10000 .224 .178 .181 .197 .224 .261.477 .175 .203 .235 .161 .348 MMP-9 A-3 1:100 .706 .626 .339 .285 3.7753.810 1.991 3.812 3.776 3.814 2.063 3.84 1:1000 .390 .162 .167 .176 .3942.157 .272 2.281 .448 1.332 .448 2.34 1:10000 .123 .142 .151 .158 .159.631 .159 .267 .167 .209 .158 .296 MMP-2 A-4 1:100 .926 .273 .258 .2401.485 2.409 1.811 1.649 1.836 3.172 2.278 1.621 1:1000 .340 .162 .162.192 .650 .974 .771 .289 .7061 .286 .775 .294 1:10000 .148 .142 .150.179 .220 .257 .208 .168 .290 .313 .230 .167

The oligopeptide therapeutically effective amount may be administered tothe mammal in many different ways and may not be limited to injections.The various methods of administration are well known in the art and someof the methods are described below.

A “mammal” to be treated by the subject method may mean either a humanor non-human animal, such as mice, primates and vertebrates.

The specific diseases that would be target diseases for a treatmentusing MMP oligopeptide sequences and/or peptidomimetic are neoplasticdiseases, inflammatory diseases, coronary artery diseases, occlusivecardiovascular diseases, degenerative diseases and infectious diseases.Some examples of neoplastic diseases may be, but not limited to, cancer,lymphoma, leukemia, and brain tumor. Some examples of inflammatorydiseases may be, but not limited to, arthritis, asthma, atherosclerosis,Crohn's disease, colitis, dermatitis, lupus erythematous etc. Someexamples of infectious diseases may include, but not limited to, arebacterial, viral, fungal, mycoplasmal, certain genetic diseases andother infections.

Drug formulations suitable for these administration routes can beproduced by adding one or more pharmacologically acceptable carriers tothe agent and then treating the mixture through a routine process knownto those skilled in the art. The mode of administration includes, butnot limited to, are non-invasive peroral, topical (example transdermal),enteral, transmucosal, targeted delivery, sustained release delivery,delayed release, pulsed release and parenteral methods. Peroraladministration may be administered both in liquid and dry state.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition as an active ingredient. Subject compositions may also beadministered as a bolus, electuary, or paste.

When an oral solid drug product is prepared, oligopeptide sequence ofMMP and/or a peptidomimetic of the MMP's is mixed with an excipient(and, if necessary, one or more additives such as a binder, adisintegrant, a lubricant, a coloring agent, a sweetening agent, and aflavoring agent), and the resultant mixture is processed through aroutine method, to thereby produce an oral solid drug product such astablets, coated tablets, granules, powder, or capsules. Additives may bethose generally employed in the art. Examples of the excipient includelactate, sucrose, sodium chloride, glucose, starch, calcium carbonate,kaolin, microcrystalline cellulose, and silicic acid; examples of thebinder include water, ethanol, propanol, simple syrup, glucose solution,starch solution, liquefied gelatin, carboxymethylcellulose,hydroxypropylcellulose, hydroxypropyl starch, methyl cellulose, ethylcellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone;examples of the disintegrant include dried starch, sodium arginate,powdered agar, sodium hydrogencarbonate, calcium carbonate, sodiumlauryl sulfate, monoglyceryl stearate, and lactose; examples of thelubricant include purified talc, stearic acid salts, borax, andpolyethylene glycol; and examples of the sweetening agent includesucrose, orange peel, citric acid, and tartaric acid.

When a liquid drug product for oral administration is prepared,oligopeptide sequence of MMP and/or a peptidomimetic of MMP's is mixedwith an additive such as a sweetening agent, a buffer, a stabilizer, ora flavoring agent, and the resultant mixture is processed through aroutine method, to thereby produce an orally administered liquid drugproduct such as an internal solution medicine, syrup, or elixir.Examples of the sweetening agent include vanillin; examples of thebuffer include sodium citrate; and examples of the stabilizer includetragacanth, acacia, and gelatin.

For purposes of transdermal (e.g., topical) administration, dilutesterile, aqueous or partially aqueous solutions (usually in about 0.1%to 5% concentration), otherwise similar to the above parenteralsolutions, may be prepared.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax, or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the appropriate body cavity and release theencapsulated compound(s) and composition(s). Formulations which aresuitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams, or spray formulations containing suchcarriers as are known in the art to be appropriate.

A targeted release portion can be added to the extended release systemby means of either applying an immediate release layer on top of theextended release core; using coating or compression processes or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

When used with respect to a pharmaceutical composition or othermaterial, the term “sustained release” is art-recognized. For example, atherapeutic composition which releases a substance over time may exhibitsustained release characteristics, in contrast to a bolus typeadministration in which the entire amount of the substance is madebiologically available at one time. For example, in particularembodiments, upon contact with body fluids including blood, spinalfluid, mucus secretions, lymph or the like, one or more of thepharmaceutically acceptable excipients may undergo gradual or delayeddegradation (e.g., through hydrolysis) with concomitant release of anymaterial incorporated therein, e.g., an therapeutic and/or biologicallyactive salt and/or composition, for a sustained or extended period (ascompared to the release from a bolus). This release may result inprolonged delivery of therapeutically effective amounts of any of thetherapeutic agents disclosed herein.

Current efforts in the area of drug delivery include the development oftargeted delivery in which the drug is only active in the target area ofthe body (for example, in cancerous tissues) and sustained releaseformulations in which the drug is released over a period of time in acontrolled manner from a formulation. Types of sustained releaseformulations include liposomes, drug loaded biodegradable microspheresand drug polymer conjugates.

Delayed release dosage formulations are created by coating a soliddosage form with a film of a polymer which is insoluble in the acidenvironment of the stomach, but soluble in the neutral environment ofthe small intestines. The delayed release dosage units can be prepared,for example, by coating a drug or a drug-containing composition with aselected coating material. The drug-containing composition may be atablet for incorporation into a capsule, a tablet for use as an innercore in a “coated core” dosage form, or a plurality of drug-containingbeads, particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Alternatively, a delayed release tablet may be formulated bydispersing tire drug within a matrix of a suitable material such as ahydrophilic polymer or a fatty compound. Suitable hydrophilic polymersinclude, but are not limited to, polymers or copolymers of cellulose,cellulose ester, acrylic acid, methacrylic acid, methyl acrylate, ethylacrylate, and vinyl or enzymatically degradable polymers or copolymersas described above. These hydrophilic polymers are particularly usefulfor providing a delayed release matrix. Fatty compounds for use as amatrix material include, but are not limited to, waxes (e.g. carnaubawax) and glycerol tristearate. Once the active ingredient is mixed withthe matrix material, the mixture can be compressed into tablets.

A pulsed release-dosage is one that mimics a multiple dosing profilewithout repeated dosing and typically allows at least a twofoldreduction in dosing frequency as compared to the drug presented as aconventional dosage form (e.g., as a solution or prompt drug-releasing,conventional solid dosage form). A pulsed release profile ischaracterized by a time period of no release (lag time) or reducedrelease followed by rapid drug release.

The phrases “parenteral administration” and “administered parenterally”as used herein refer to modes of administration other than enteral andtopical administration, such as injections, and include withoutlimitation intravenous, intramuscular, intrapleural, intravascular,intrapericardial, intra-arterial, intrathecal, intracapsular,intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid,intraspinal and intrasternal injection and infusion.

Certain pharmaceutical compositions disclosed herein suitable forparenteral administration comprise one or more subject compositions incombination with one or more pharmaceutically acceptable sterile,isotonic, aqueous, or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic within the blood of the intended recipient orsuspending or thickening agents.

When an injection product is prepared, oligopeptide sequence of MMPand/or a peptidomimetic of MMP's is mixed with an additive such as a pHregulator, a buffer, a stabilizer, an isotonicity agent, or a localanesthetic, and the resultant mixture is processed through a routinemethod, to thereby produce an injection for subcutaneous injection,intramuscular injection, or intravenous injection. Examples of the pHregulator or buffer include sodium citrate, sodium acetate, and sodiumphosphate; examples of the stabilizer include sodium pyrosulfite, EDTA,thioglycollic acid, and thiolactic acid; examples of the localanesthetic include procaine hydrochloride and lidocaine hydrochloride;and examples of the isotonicity agent include sodium chloride andglucose.

Adjuvants are used to enhance the immune response. Various types ofadjuvants are available. Haptens are used as adjuvants in thisdisclosure. Freund's adjuvants may also be used to produce water-in-oilemulsions of immunogens. Antigens in water-in-oil emulsions stimulatehigh and long-lasting antibody responses which can be attributed to theslow release of antigen. Antigens (preferably in saline) are typicallymixed with an equal volume of the adjuvant to form an emulsion.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are within the scope of soundmedical judgment, suitable for use in contact with the tissues ofmammals, human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,solvent or encapsulating material involved in carrying or transportingany subject composition, from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of a subjectcomposition and not injurious to the patient. In certain embodiments, apharmaceutically acceptable carrier is non-pyrogenic. Some examples ofmaterials which may serve as pharmaceutically acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

In certain embodiments, the pharmaceutical compositions described hereinare formulated in a manner such that said compositions will be deliveredto a mammal in a therapeutically effective amount, as part of aprophylactic, preventive or therapeutic treatment.

In certain embodiments, the dosage of the oligopeptide compositions,which may be referred as therapeutic composition provided herein may bedetermined by reference to the plasma concentrations of the therapeuticcomposition or other encapsulated materials. For example, the bloodsamples may be tested for their immune response to their correspondingoligopeptides.

The therapeutic compositions provided by this application may beadministered to a subject in need of treatment by a variety ofconventional routes of administration, including orally, topically,parenterally, e.g., intravenously, subcutaneously or intramedullary.Further, the therapeutic compositions may be administered intranasally,as a rectal suppository, or using a “flash” formulation, i.e., allowingthe medication to dissolve in the mouth without the need to use water.Furthermore, the compositions may be administered to a subject in needof treatment by controlled release dosage forms, site specific drugdelivery, transdermal drug delivery, patch (active/passive) mediateddrug delivery, by stereotactic injection, or in nanoparticles.

Expressed in terms of concentration, an active ingredient can be presentin the therapeutic compositions of the present invention for localizeduse about the cutis, intranasally, pharyngolaryngeally, bronchially,intravaginally, rectally, or ocularly.

For use as aerosols, the active ingredients can be packaged in apressurized aerosol container together with a gaseous or liquefiedpropellant, for example, dichlorodifluoromethane, carbon dioxide,nitrogen, propane, and the like, with the usual adjuvants such ascosolvents and wetting agents, as may be necessary or desirable.

The most common routes of administration also include the preferredtransmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) andinhalation routes.

In addition, in certain embodiments, subject compositions of the presentapplication maybe lyophilized or subjected to another appropriate dryingtechnique such as spray drying. The subject compositions may beadministered once, or may be divided into a number of smaller doses tobe administered at varying intervals of time, depending in part on therelease rate of the compositions and the desired dosage.

Formulations useful in the methods provided herein include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal, aerosol and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof a subject composition which may be combined with a carrier materialto produce a single dose may vary depending upon the subject beingtreated, and the particular mode of administration.

The therapeutically acceptable amount described herein may beadministered in inhalant or aerosol formulations. The inhalant oraerosol formulations may comprise one or more agents, such as adjuvants,diagnostic agents, imaging agents, or therapeutic agents useful ininhalation therapy. The final aerosol formulation may for examplecontain 0.005-90% w/w, for instance 0.005-50%, 0.005-5% w/w, or0.01-1.0% w/w, of medicament relative to the total weight of theformulation.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

The therapeutic acceptable dosage may be combined with other drugs andmay be treated as a combination drug.

In FIG. 2, the produced antibodies 241 are checked using ELISA.Innoculating the mice (228) was done using cancerous cells 246 byinjection. The block of MMP's 230 by antigen 248 was observed. IntactECM 234, shown as 250 by inhibiting ECM destruction 232 was demonstratedby in vivo and in vitro studies as discussed above and below.

Cancer Cell Lines and Culture

Human cervical cancer (Hela) and murine melanoma B16FO cells obtainedfrom ATCC (American Type Culture Collection, Rockville, Md.) weremaintained in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum and antibiotics. The media and sera used wereobtained from ATCC, and antibiotics (penicillin and streptomycin) werefrom Gibco BRL, Long Island, N.Y.

Matrigel™ Invasion

Invasion studies were conducted using Matrigel™ (Becton Dickinson)inserts in 24-well plates. Suspended in medium containing anti-sera fromthe experimental immunized mice 1:100 dilution, human cervical cancerHeLa cells seeded on the insert in the well. The well has human dermalfibroblast primed media as chemotaxtant. The plates with the insertswere then incubated in a culture incubator equilibrated with 95% air and5% CO: for 24 hours. After incubation, the media from the wells werewithdrawn. The cells on the upper surface of the insert were gentlyscrubbed away with cotton swabs. The cells that had penetrated theMatrigel™ membrane were stained with hematoxylin and eosin (H&E) andvisually counted under the microscope.

The inhibition was compared to the control. FIGS. 3A-3D show variousmatrix metalloproteinase oligopeptide and their effectiveness. There aremany cells that are invading through the Matrigel™ compared to MMP-9A1-3 and MMP-2 A4 exposed cells. For example, in FIG. 3A, cells numberedas 1001A-1003A are more in number compared to MMP-9 A1 treated cells1004 A-1005A. Similar results are found for MMP-9 2-3 and MMP-2 A4 insubsequent figures.

The in vivo studies described above presented some interesting resultsin the form of weight of the mice, tumor weight, tumor burden studiesand effectiveness of various oligopeptides used in a therapeuticallyeffective amount to inhibit ECM digestion or destruction.

After dosing the mice, on 45^(th) day, mice were bleed through orbitalpuncture and blood was collected in 2-ml micro centrifuged tube. Aftertesting for immune response against individual peptides, the mice ineach group were inoculated subcutaneously with 0.5×10⁶ in 0.2 ml PBS.After injection, the mice were returned the cages and fed regular Purinamouse chow diet. After four weeks, the mice were sacrificed and theirtumors were excised. Dimensions (length and width) of tumors weremeasured using digital caliper, and tumor burden was calculated usingthe following formula: 0.5×length×width.

The weight of the animals did not significantly change before and aftertreatment as shown in FIG. 4. FIG. 5 shows the therapeutic effect of allmatrix metalloproteinase oligopeptide immunization effect on tumorweight. FIG. 6 shows the significant effect of tumor volume as comparedto control tumors, further proving the effectiveness of the treatmentmethod. FIG. 7 A-E visually shows some of the tumors excised anddisplayed. The significant reduction in size is very apparent. In one ofthe MMP-9 A 3 one of the tumors have completely disappeared. FIG. 8shows a three dimensional structure of the MMP molecule. The figure alsodiscloses the region of the molecule that enables MMP-9 A1, MMP-9 A2 andMMP-9 A3 in as a three dimensional structure. Peptidomimetic of theseMMP may be designed based on the structure to inhibit MMP expression andprevent metastasis.

In addition, it will be appreciated that the various sequences,immunization processes, and methods of treatment disclosed herein may beembodied using means for achieving the various combinations oftherapeutic dosage and delivery methods to treat a specific disease.Accordingly, the specification and drawings are to be regarded in anillustrative rather than a restrictive sense.

1. A peptide consisting of the amino acid sequence of SEQ ID NO:
 2. 2. Apeptide consisting of the amino acid sequence of SEQ ID NO:
 3. 3. Apeptide consisting of the amino acid sequence of SEQ ID NO:
 4. 4. Thepeptide of claim 1, 2, or 3, which is biotinylated.
 5. A conjugatecomprising the peptide of claim 1, 2, or 3 covalently joined to keyholelimpet hemocyanin (KLH).
 6. A pharmaceutical composition comprising oneor more of the peptides of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4and a pharmaceutically acceptable carrier.
 7. The pharmaceuticalcomposition of claim 6, wherein said one or more peptides is covalentlyjoined to keyhole limpet hemocyanin (KLH).
 8. The pharmaceuticalcomposition of claim 6 further comprising an adjuvant.
 9. A method ofinducing an immune response against human matrix metalloproteinase-9(MMP-9) in a mammalian subject, said method comprising administering tosaid subject an effective amount of a composition comprising one or moreof the peptides of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 toelicit the production of antibodies against MMP-9.
 10. The method ofclaim 9, wherein said antibodies are effective to block the enzymaticactivity of MMP-9 and inhibit tumor cell invasion.
 11. The method ofclaim 9, wherein said mammalian subject is a human.
 12. The method ofclaim 9, wherein said mammalian subject has an invasive tumor.
 13. Themethod of claim 12, wherein the tumor burden of the mammalian subject isreduced.
 14. A method for suppressing the growth of an invasive tumor ina mammalian subject, said method comprising administering to saidsubject an effective amount of the composition of claim 4 to elicitantibodies against human matrix metalloproteinase-9 (MMP-9) in thesubject, wherein said antibodies are effective to block the enzymaticactivity of MMP-9 and inhibit tumor cell invasion, thereby suppressingthe growth of the tumor in the subject.
 15. The method of claim 14,wherein the tumor burden of the mammalian subject is reduced.